Human Immunodeficiency virus (HIV) is the etiologic agent of AIDS. The pathogenesis of HIV -induced disease is complex and multifactoral. Several key HIV and cellular proteins have been assigned to be necessary for the course of infection including the transactivator Tat. Viral clones deficient in Tat will not replicate in vitro or in vivo to high titers. The current proposal aims to extend our previous results, which were obtained during the past four years on the effect of Tat peptide derivatives in inhibition of HIV-1. Previous specific aims were successfully met in a timely manner, and they included determination of the target(s) of the Tat peptide inhibitor, defining the minimum structural requirement for the inhibitoryactivity of the Tat core peptide derivative, and determining the range of inhibition on a few HIV-1 isolates. More preliminary data is included to show the target of these Tat peptide inhibitors using in vitro and in vivo assays. Tat peptides were also cyclized at small scale and used in non-specific toxicity assays as well as bioavailability assays in animals with great success. Therefore, we have proposed to continue our initial goal, which was to develop HIV-1 Tat inhibitors that are specific to the Tat/TAR complex. We will optimize peptide cyclization of the short Tat peptide by using new cyclization strategies includingthe use of Cys 4 and Cys 2 compounds. Tat peptides will also contain either Tat or SV40 NLS sequences for efficient delivery. Biological inhibitory activities in addition various biochemical and virological methods will be utilized. To define the breath of Tat peptide inhibition, we will determine the inhibitoryactivity of the Tat peptide on various HIV- 1 isolates including drug-resistant strains. We will assess the cytotoxicity of Tat peptides on replicating T-cells and PBMCs as well as determining the inhibitory activity of these compounds on various HIV-1 wild type, reverse transcriptase and protease resistant viruses with different tropisms. Also, we will utilize the SCID-Hu Thy/Liv mice model to test toxicity and viral inhibition in vivo. Finally, to define whether these inhibitors have any non-specific cellular toxicity, we will utilize gene expression profiling of Tat peptides in treated cells. This will be accomplished through the use of clustering and data analysis methods in PBMC infected cells. Validation of the data will initially include in silico prediction algorithms, independent array data sets, and functional assays using probabilistic rational models, GenMAPP, and Pubgene.